The assembly of enveloped animal viruses is characterized by selective inclusion of the viral genome and accessory viral proteins into a budding viral particle. Although the mechanisms for selective encapsidation or packaging are not well characterized, it has been postulated that the recognition of viral envelope proteins within the plasma membrane by the viral nucleocapsids represents one probable control point for packaging specificity.
Using internal image anti-idiotype antibodies, Vaux et al. (Nature (London) 336:36-42 (1988)) have shown that the nucleocapsid of Semliki Forest virus (SFV) contains a specific receptor for the cytoplasmic tail of the virion E2 spike glycoprotein. Vaux et al. suggested that a specific receptor-ligand-like interaction between the two is likely to be critical in the organization of the budding of SFV and related viruses from infected cells.
In apparent contrast to the high degree of specificity of SFV for the E2 protein, is the well-known phenomenon of "pseudotype" formation, in which mixed infection of a cell by one virus and retroviruses results in the production of progeny virions bearing the genome of one of the viruses encapsidated by the envelope proteins of the other. These phenotypically mixed viruses form plaques on appropriate indicator cells and can be neutralized by sera raised against the specific envelope protein. One virus known to participate in pseudotype formation is vesicular stomatitis virus (VSV), a member of the rhabdovirus family.
The mechanism for the inclusion of the envelope protein of one virus into the virions of an unrelated virus is uncertain. Sequence comparison of VSV G protein and retrovirus envelope proteins reveals no significant sequence similarity among these proteins. Heretofore, it has also been difficult to determine whether G protein alone in the absence of other VSV-encoded proteins can participate in pseudotype formation. Pseudotypes do not form between-VSV and alphaviruses such as SFV even though pseudotypes can form between two alphaviruses or between alphaviruses and related flaviviruses such as Japanese encephalitis virus.
In some cases, phenotypic mixing is unilateral, as in the case of VSV with fowl plaque virus (FPV) or VSV with Sindbis virus. The pseudotype virus particle VSV(FPV) containing the VSV genome encapsidated by the envelope protein of FPV and the pseudotype virus particle VSV(Sindbis) have been demonstrated, but the reverse pseudotypes, FPV(VSV) and Sindbis(VSV), containing FPV or Sindbis virus genome with the VSV G protein, have not been detected.
Mixed infection of cells with retroviruses and VSV usually results in the formation of pseudotypes with much lower titers than that of VSV generated from the same cells. It is not clear whether this is due to the specificity of the interaction between the retroviral nucleocapsid and the G protein or due to other factors.
Retroviral vectors have been used to transfer genes efficiently by exploiting the viral infectious process. Foreign genes cloned into the retroviral genome can be delivered efficiently to cells susceptible to infection by the retrovirus. Through other genetic manipulations, the replicative capacity of the retroviral genome can be destroyed. The vectors introduce new genetic material to a cell but are unable to replicate. A helper virus or a packaging system can be used to permit vector particle assembly and egress. As used herein, the term "vector particle" refers to viral-like particles that are capable of introducing nucleic acid into a cell through a viral-like entry mechanism. Such vector particles, can under certain circumstances, mediate the transfer of genes into the cells they infect.
It is possible to alter the range of cells that these vectors can infect by including an envelope gene from another closely related virus. Miller et al. (Mol. Cell. Biol. 5:431-437 (1985)) constructed a MoMLV-derived vector to introduce a selectable marker, dihydrofolate reductase, into susceptible cells, and included the envelope region from the related amphotropic retrovirus 4070A to broaden the host range of the vector. Other investigators have described pseudotypes of retroviral vectors whose host cell range has been altered by substitution of envelope proteins from different viruses. Substitution of the gibbon ape leukemia virus envelope protein for the amphotropic retroviral envelope has resulted in vectors capable of infecting bovine and hamster cells, species not susceptible to infection with retroviral vectors containing the MoMLV envelope protein (Miller et al, J. Virol., 65:2220-2224 (1991)). Similarly, substitution of the HTLV I envelope protein has been shown to restrict the host cell range of a MoMLV-based vector to cells infectable by HTLV I (Wilson et al., J. Virol., 63:2374-2378, (1989)).
Retroviral vectors derived from Moloney murine leukemia virus (MoMLV) are important tools for stable gene transfer into mammalian cells. They have been used to study gene regulation and expression and to facilitate gene transfer for studies of human gene therapy. Two significant limitations to the use of these retroviral vectors are the restricted host cell range and the inability to produce high-titer virus. Infection with retroviral vectors results from specific interaction of the viral envelope glycoprotein with cellular receptors, defining the host range and determining the efficiency of infection. Attempts to concentrate retroviral vectors by centrifugation or other physical means generally result in loss of infectious virus with only minimal increases in titer. The instability of retroviral particles may be related to structural characteristics of the envelope protein and modification of envelope components might, therefore, result in a more stable particle.
As stated above, it is not clear what signals are required to direct the functional assembly of the vector particle, nor is it known what factors permit the nucleocapsid and the membrane-associated protein to interact and complete packaging. Accordingly, heretofore, alterations in the host range have not been effected by including heterologous membrane-associated proteins within a vector particle. By "heterologous membrane-associated protein" it is meant a membrane-associated protein having at least one origin other than a virus of the same viral family as the origin of the nucleocapsid protein of the vector particle. As used herein, viral "family" shall be used to refer to the taxonomic rank of family, as assigned by the International Committee on Taxonomy of Viruses.